Oil crop genetic modification for producing added value lipids.

Plant lipids, mainly stored in seeds and other plant parts, are not only a crucial resource for food and fodder but are also a promising alternative to fossil oils as a chemical industry feedstock. Oil crop cultivation and processing are always important parts of agriculture worldwide. Vegetable oils containing polyunsaturated fatty acids, very long chain fatty acids, conjugated fatty acids, hydroxy fatty acids and wax esters, have outstanding nutritional, lubricating, surfactant, and artificial-fibre-synthesis properties, amongst others. Enhancing the production of such specific lipid components is of economic interest. There has been a considerable amount of information reported about plant lipid biosynthesis, including identification of the pathway map of carbon flux, key enzymes (and the coding genes), and substrate affinities. Plant lipid biosynthesis engineering to produce special oil compounds has become feasible, although until now, only limited progress has been made in the laboratory. It is relatively easy to achieve the experimental objectives, for example, accumulating novel lipid compounds in given plant tissues facilitated by genetic modification. Applying such technologies to agricultural production is difficult, and the challenge is to make engineered crops economically attractive, which is impeded by only moderate success. To achieve this goal, more complicated and systematic strategies should be developed and discussed based on the relevant results currently available.

Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica.

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.